Preparation Of Aptamer Functional Materials For Selective Adsorption Of Mycotoxins And Its Application In Analysis Of Dairy Products

In recent decades,aflatoxin and ochratoxin in food have attracted more and more attention due to severe toxicity and wide range of distribution.ELISA,HPLC and HPLC-MS analysis techniques are widely used in the detection of aflatoxin and ochratoxin.Due to the complex food matrix and the low content of mycotoxins,pre-treatment methods for enriching and separating mycotoxins from food samples are necessary（such as solid phase extraction）.However,current solid phase extraction materials available on the market,including HLB columns and C18monolithic columns limit adsorption selectivity and enrichment efficiency.Other commonly used immunoaffinity chromatography materials are often expensive,easily inactivated,non-reusable as well as complex to synthesize and modify.Therefore,the development of novel adsorption materials of mycotoxin has become a research hotspot.Recently,many studies have shown that aptamer can specifically bind to specific target substances.In this paper,the selected aptamers could interact with ochratoxin A（OTA）and aflatoxin M1（AFM₁）,respectively.Aptamers of OTA and AFM₁ were respectively modified on the carrier material to prepare two kinds of novel aptamer functional materials.Their adsorption properties and enrichment conditions for OTA and AFM₁ were studied separately.And the aptamer functional material is applied to the detection of AFM₁ and its analogues in dairy products.The main research contents with its results are as follows:1.The colorimetric determination conditions of aptamers and gold nanoparticles（GNPs）were carried out by using GNPs colorimetry.With addition of OTA and AFM₁,the interaction between the aptamers and target（OTA or AFM₁）were characterized thought the changes of A650/A530 at 650 nm and 530 nm.The results indicated that two kinds of aptamers could respectively specifically bind to OTA and AFM₁ to produce color change in the GNPs system,which indicate that strong specific binding affinity between aptamer and target.Thus,two kinds of aptamers could be used as functional monomers to prepare specific affinity materials.2.The preparation of Aptamer-1 functionalized magnetic beads is through the biological recognition of streptavidin and biotin,and its modification conditions and adsorption properties were studied.The analysis of the Aptamer-1 on the magnetic beads was carried out by UPLC-Q-Orbitrap MS/MS,and the optimization conditions of synthesis were as follows:the concentration of Aptamer-1 was 0.5μmol/L,and modified in pH 7.2 20 mmol/L Tris-HCl buffer for 4 h.The optimization of the enrichment and separation conditions of Aptamer-1 functionalized magnetic beads for OTA were shown as follow:adsorption time was 120min,the concentration of Mg²⁺was 10 mmol/L,the concentration of K⁺was 5 mmol/L,temperature was 50°C,and eluted by sonicating for 10min with 10%acetonitrile-water（v/v）.The adsorption properties experimental results showed that Aptamer-1 functionalized magnetic beads had high batch stability and time stability and had only a high specific affinity for OTA with the adsorption capacity of 38.67μg/g.3.The preparation of Aptamer-2 functionalized microspheres was characterized by EDC/NHs-mediated amidation,then the modification was characterized and the adsorption properties was studied.UPLC-Q-Orbitrap MS/MS was used to characterize the modification of Aptamer-2 on microspheres,and the results showed that Aptamer-2 could completely modify on the surface of carboxyl microspheres.The optimization of the enrichment and separation conditions of Aptamer-2functionalized microspheres for AFM₁ were shown as follow:no buffer system was required and the extraction pH was 7,the concentration of Mg²⁺was 10 mmol/L,and the elution solvent was 75%organic solvent（methanol or acetonitrile）.The adsorption properties experimental results showed that Aptamer-2 functionalized microspheres had good adsorption selectivity for AFM₁ and its structural analogues which represented a derivative of dihydrofuranoxaphthalene containing a bisfuran ring moiety and an oxaphthaleneketo group.And it had high adsorption capacity（233.1μg/g）,high batch stability and high time stability.4.Based on solid phase extraction by Aptamer-2 functionalized microspheres,an analytical method for the determination of AFM₁ and its analogues（AFB₁,AFB₂,AFG₁,AFG₂）in dairy products was established and a series of methodological studies were completed.The method for the determination AFM₁ and its analogues in dairy products demonstrated a good linear correlation in the range of 0 to 2.0μg/L（R²≥0.9985）,low matrix effect and high detection sensitivity（detection limits of 7 ng/kg in milk and 21 ng/kg in milk power）.Furthermore,excellent spike recovery（86.72¹09.9%）and good repeatability（RSD 2.6⁸.7%）were obtained in the spike recovery experiment of both blank and detected samples.It provides an analytical method for the selective adsorption of AFM₁ and its analogues exhibiting a dihydrofuranoxaphthalene scaffold in dairy products with excellent accuracy and repeatability.